Yeast expression systems can be used to effectively produce proteins, such as enzymes, hormones, and vaccine proteins, in part, because some yeast grow rapidly to high cell densities, are grown in simple and inexpensive media, and are eukaryotes so they can modify proteins in a manner similar to native proteins in mammals. Additionally, with a proper signal sequence, the expressed protein can be secreted into the culture medium for convenient isolation and purification. Some yeast expression systems are also accepted in the food and pharmaceutical industries as being safe for the production of pharmaceuticals and food products, unlike fungal and bacterial expression systems which may in some cases be unsafe, for example, for human food manufacturing.
Thus, it is beneficial for a variety of industries, such as the food and animal feed industries, the human and animal health industries, and the like, to develop or improve yeast expression systems that can be used to express high levels of proteins to increase yield, reduce the expense of isolation and purification of proteins, and reduce the costs of human and animal health products and food products.
A variety of types of yeast expression systems have been developed involving either the use of inducible or constitutive expression of proteins using nucleic acids encoding homologous or heterologous proteins, under the control of a yeast promoter. Promoters are regulatory elements that are linked to the 5′ end of a nucleic acid encoding a protein, and may interact with various regulatory factors in the host cell (e.g., a yeast host cell) to control transcription of RNA from DNA. Promoters may also control the timing of transcription of RNA from DNA. For example, the AOX 1 promoter has been identified in the yeast Pichia pastoris, and is commonly used in yeast expression systems because it is a tightly regulated, strong promoter.
Due to the importance of yeast expression systems for a variety of industries, including the human pharmaceuticals industry, and the human food and animal feed industries, the improvement of yeast expression systems is the focus of much research and development. Accordingly, the present inventors have identified promoters from Pichia pastoris that are particularly effective for use in expression of proteins in yeast. The promoters described herein can be used, for example, in methanol-inducible yeast expression systems, or in expression systems for the constitutive expression of proteins.
In one illustrative embodiment of the invention, an isolated nucleic acid is provided wherein the sequence of the isolated nucleic acid comprises a sequence, for example, at least 90%, 95%, or 98% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 as described herein, or at least 90%, 95%, or 98% identical to a fragment thereof, wherein the isolated nucleic acid comprises the sequence of a methanol-inducible Pichia pastoris promoter. In other embodiments, expression vectors, host cells, and DNA constructs comprising these promoter sequences are provided.
In another embodiment, a method of producing a protein using these promoter sequences is provided. The method comprises the steps of culturing in a culture medium a host cell comprising a first expression cassette comprising any of the above promoter sequences operably linked to a heterologous coding sequence encoding a protein, wherein the culturing is done under conditions permitting expression of the protein. In another illustrative embodiment, an isolated protein produced according to this method is provided.
In one illustrative embodiment of the invention, an isolated nucleic acid is provided wherein the sequence of the isolated nucleic acid comprises a sequence, for example, at least 90%, 95%, or 98% identical to a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14 as described herein, or at least 90%, 95%, or 98% identical to a fragment thereof, wherein the isolated nucleic acid comprises the sequence of a constitutive Pichia pastoris promoter. In other embodiments, expression vectors, host cells, and DNA constructs comprising these promoter sequences are provided.
In another embodiment, a method of producing a protein using these promoter sequences is provided. The method comprises the steps of culturing in a culture medium a host cell comprising a first expression cassette comprising any of the above promoter sequences operably linked to a heterologous coding sequence encoding a protein, wherein the culturing is done under conditions permitting expression of the protein. In another illustrative embodiment, an isolated protein produced according to this method is provided.
All of the embodiments described in the following clause list are also contemplated for use in accordance with the invention. For all of the embodiments described in the following clauses, any applicable combination of embodiments is considered to be in accordance with the invention.
1. An isolated nucleic acid wherein the sequence of the isolated nucleic acid comprises a sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or at least 90% identical to a fragment thereof, wherein the isolated nucleic acid comprises the sequence of a methanol-inducible Pichia pastoris promoter.
2. The isolated nucleic acid of clause 1 wherein the sequence of the isolated nucleic acid is at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or at least 95% identical to a fragment thereof.
3. The isolated nucleic acid of clause 1 wherein the sequence of the isolated nucleic acid is at least 98% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or at least 98% identical to a fragment thereof.
4. The isolated nucleic acid sequence of clause 1 wherein the sequence of the isolated nucleic acid is a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or a fragment thereof.
5. The isolated nucleic acid of any one of clauses 1 to 4 operably linked to a heterologous coding sequence.
6. The isolated nucleic acid of clause 5 wherein the heterologous coding sequence encodes a protein selected from the group consisting of a toxin, an antibody, a hormone, an enzyme, a growth factor, a cytokine, a structural protein, an immunogenic protein, and a cell signaling protein.
7. The isolated nucleic acid of clause 6 wherein the protein is an enzyme for use in animal feed.
8. The isolated nucleic acid of clause 7 wherein the protein is selected from the group consisting of a phytase, a mannanase, a galactosidase, an amylase, a glucanase, a protease, a cellulase, and a xylanase.
9. The isolated nucleic acid of clause 8 wherein the protein is a phytase.
10. The isolated nucleic acid of clause 8 wherein the protein is a galactosidase.
11. An expression vector comprising the isolated nucleic acid of any one of clauses 1 to 10.
12. A host cell comprising the expression vector of clause 11.
13. A host cell comprising the isolated nucleic acid of any one of clauses 1 to 10.
14. The host cell of any one of clauses 12 or 13 wherein the host cell is a Pichia species.
15. The host cell of clause 14 wherein the Pichia species is Pichia pastoris. 
16. A DNA construct comprising the isolated nucleic acid of any one of clauses 1 to 10.
17. A method of producing a protein, the method comprising the step of
culturing in a culture medium a host cell comprising a first expression cassette comprising the isolated nucleic acid of any one of clauses 1 to 4 operably linked to a heterologous coding sequence encoding a protein, wherein the culturing is done under conditions permitting expression of the protein.
18. The method of clause 17 wherein the protein is selected from the group consisting of a toxin, an antibody, a hormone, an enzyme, a growth factor, a cytokine, a structural protein, an immunogenic protein, and a cell signaling protein.
19. The method of clause 18 wherein the protein is an enzyme for use in animal feed.
20. The method of clause 19 wherein the protein is selected from the group consisting of a phytase, a mannanase, a galactosidase, an amylase, a glucanase, a cellulase, a protease, and a xylanase.
21. The method of clause 20 wherein the protein is a phytase.
22. The method of clause 20 wherein the protein is a galactosidase.
23. The method of any one of clauses 17 to 22 wherein the protein is expressed using the first expression cassette in combination with a second expression cassette.
24. The method of clause 23 wherein the second expression cassette comprises the heterologous coding sequence encoding the protein operably linked to an isolated nucleic acid having a sequence comprising the sequence of SEQ ID NO:15 or SEQ ID NO:16 wherein SEQ ID NO:15 and SEQ ID NO:16 have promoter activity, or any other AOX 1 or AOX 2 promoter sequence.
25. The method of clause 24 wherein the protein is expressed using the first expression cassette, the second expression cassette, and a third expression cassette.
26. The method of clause 25 wherein the third expression cassette comprises the heterologous coding sequence encoding the protein operably linked to an isolated nucleic acid having a sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or at least 90% identical to a fragment thereof.
27. The method of clause 23 wherein the second expression cassette comprises the heterologous coding sequence encoding the protein operably linked to an isolated nucleic acid having a sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or at least 90% identical to a fragment thereof.
28. An isolated protein produced according to the method of any one of clauses 17 to 27.
29. An isolated nucleic acid wherein the sequence of the isolated nucleic acid comprises a sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, or at least 90% identical to a fragment thereof, wherein the nucleic acid comprises the sequence of a constitutive Pichia pastoris promoter.
30. The isolated nucleic acid of clause 29 wherein the sequence of the isolated nucleic acid is at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14 or at least 95% identical to a fragment thereof.
31. The isolated nucleic acid of clause 29 wherein the sequence of the isolated nucleic acid is at least 98% identical to a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14 or at least 98% identical to a fragment thereof.
32. The isolated nucleic acid sequence of clause 29 wherein the sequence of the isolated nucleic acid is a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, or a fragment thereof.
33. The isolated nucleic acid of any one of clauses 29 to 32 operably linked to a heterologous coding sequence.
34. The isolated nucleic acid of clause 33 wherein the heterologous coding sequence encodes a protein selected from the group consisting of a toxin, an antibody, a hormone, an enzyme, a growth factor, a cytokine, a structural protein, an immunogenic protein, and a cell signaling protein.
35. The isolated nucleic acid of clause 34 wherein the protein is an enzyme for use in animal feed.
36. The isolated nucleic acid of clause 35 wherein the protein is selected from the group consisting of a phytase, a mannanase, a galactosidase, an amylase, a glucanase, a cellulase, a protease, and a xylanase.
37. The isolated nucleic acid of clause 36 wherein the protein is a phytase.
38. The isolated nucleic acid of clause 36 wherein the protein is a galactosidase.
39. An expression vector comprising the isolated nucleic acid of any one of clauses 29 to 38.
40. A host cell comprising the expression vector of clause 39.
41. A host cell comprising the isolated nucleic acid of any one of clauses 29 to 38.
42. The host cell of any one of clauses 40 or 41 wherein the host cell is a Pichia species.
43. The host cell of clause 42 wherein the Pichia species is Pichia pastoris. 
44. A DNA construct comprising the isolated nucleic acid of any one of clauses 29 to 38.
45. A method of producing a protein, the method comprising the step of culturing in a culture medium a host cell comprising a first expression cassette comprising the isolated nucleic acid of any one of clauses 29 to 38 operably linked to a heterologous coding sequence encoding a protein, wherein the culturing is done under conditions permitting expression of the protein.
46. The method of clause 45 wherein the protein is selected from the group consisting of a toxin, an antibody, a hormone, an enzyme, a growth factor, a cytokine, a structural protein, an immunogenic protein, and a cell signaling protein.
47. The method of clause 46 wherein the protein is an enzyme for use in animal feed.
48. The method of clause 47 wherein the protein is selected from the group consisting of a phytase, a mannanase, a galactosidase, an amylase, a glucanase, a cellulase, a protease, and a xylanase.
49. The method of clause 48 wherein the protein is a phytase.
50. The method of clause 48 wherein the protein is a galactosidase.
51. The method of any one of clauses 45 to 50 wherein the protein is expressed using the first expression cassette in combination with a second expression cassette.
52. The method of clause 51 wherein the second expression cassette comprises the heterologous coding sequence encoding the protein operably linked to an isolated nucleic acid having a sequence comprising the sequence of SEQ ID NO:15 or SEQ ID NO:16 wherein SEQ ID NO:15 and SEQ ID NO:16 have promoter activity, or any other AOX 1 or AOX 2 promoter sequence.
53. The method of clause 51 wherein the protein is expressed using the first expression cassette, the second expression cassette, and a third expression cassette.
54. The method of clause 53 wherein the third expression cassette comprises the heterologous coding sequence encoding the protein operably linked to an isolated nucleic acid having a sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or at least 90% identical to a fragment thereof.
55. The method of clause 51 wherein the second expression cassette comprises the heterologous coding sequence encoding the protein operably linked to an isolated nucleic acid having a sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or at least 90% identical to a fragment thereof.
56. An isolated protein produced according to the method of any one of clauses 45 to 55.
57. The host cell of any one of clauses 40 or 41 wherein the host cell is selected from the group consisting of Hansenula species, Pichia species, Saccharomyces species, Schizosaccharomyces species, Torulaspora species, a Candida species, a Yarrowia species, and Kluveromyces species.
58. A host cell comprising the DNA construct of clause 44 wherein the host cell is selected from the group consisting of Hansenula species, Pichia species, Saccharomyces species, Schizosaccharomyces species, Torulaspora species, a Candida species, a Yarrowia species, and Kluveromyces species.
59. The method of any one of clauses 45 to 55 wherein the host cell is selected from the group consisting of Hansenula species, Pichia species, Saccharomyces species, Schizosaccharomyces species, a Yarrowia species, Torulaspora species, Candida species, and Kluveromyces species.
60. The host cell of any one of clauses 12 or 13 wherein the host cell is a methylotrophic yeast.
61. The host cell of clause 60 wherein the host cell is selected from the group consisting of Hansenula species, Pichia species, and Candida species.
62. A host cell comprising the DNA construct of clause 16 wherein the host cell is a methylotrophic yeast.
63. The host cell of clause 62 selected from the group consisting of Hansenula species, Pichia species, and Candida species.
64. The method of any one of clauses 17 to 27 wherein the host cell is a methylotrophic yeast.
65. The method of clause 64 wherein the host cell is selected from the group consisting of Hansenula species, Pichia species, and Candida species.
66. The method of any one of clauses 25 or 53 wherein the third expression cassette comprises the heterologous coding sequence encoding the protein operably linked to an isolated nucleic acid having a sequence of SEQ ID NO:15 or SEQ ID NO:16 wherein SEQ ID NO:15 and SEQ ID NO:16 have promoter activity, or any other AOX 1 or AOX 2 promoter sequence.
67. A method of producing one or more proteins, the method comprising the step of culturing in a culture medium a host cell comprising a first expression cassette, a second expression cassette, and one or more additional expression cassettes, wherein each of the one or more additional expression cassettes comprises the isolated nucleic acid of any one of clauses 1 to 4 operably linked to a heterologous coding sequence encoding the one or more proteins, wherein the culturing is done under conditions permitting expression of the one or more proteins.
68. A method of producing one or more proteins, the method comprising the step of
culturing in a culture medium a host cell comprising a first expression cassette, a second expression cassette, and one or more additional expression cassettes, wherein each of the one or more additional expression cassettes comprises the isolated nucleic acid of any one of clauses 29 to 38 operably linked to a heterologous coding sequence encoding the one or more proteins, wherein the culturing is done under conditions permitting expression of the one or more proteins.
69. The method of any one of clauses 17 or 45 further comprising the step of purifying the protein from the medium of the cultured host cell.
70. The method of any one of clauses 67 or 68 further comprising the step of purifying one or more of the one or more proteins from the medium of the cultured host cell.
71. The isolated nucleic acid, host cell, expression vector, isolated protein, DNA construct, or method of any one of clauses 1 to 70 wherein the isolated nucleic acid consists of any one of SEQ ID NOS. 1 to 14, or a fragment thereof.
72. An isolated nucleic acid consisting of any one of SEQ ID NOS. 1 to 14, or a fragment thereof.
73. The isolated nucleic acid of clause 5 wherein the heterologous coding sequence encodes a protein or a polypeptide selected from the group consisting of a toxin, an antibody, a hormone, an enzyme, a growth factor, a cytokine, a structural protein, an immunogenic protein, and a cell signaling protein.
74. The isolated nucleic acid of clause 29 wherein the heterologous coding sequence encodes a protein or a polypeptide selected from the group consisting of a toxin, an antibody, a hormone, an enzyme, a growth factor, a cytokine, a structural protein, an immunogenic protein, and a cell signaling protein.
75. A method of producing a protein or a polypeptide, the method comprising the step of
culturing in a culture medium a host cell comprising a first expression cassette comprising the isolated nucleic acid of any one of clauses 1 to 4 operably linked to a heterologous coding sequence encoding the protein or the polypeptide, wherein the culturing is done under conditions permitting expression of the protein or the polypeptide.
76. A method of producing a protein or a polypeptide, the method comprising the step of culturing in a culture medium a host cell comprising a first expression cassette comprising the isolated nucleic acid of any one of clauses 29 to 38 operably linked to a heterologous coding sequence encoding the protein or the polypeptide, wherein the culturing is done under conditions permitting expression of the protein or the polypeptide.